Simultaneous Analysis of Amino Acids, Acylcarnitines, and ...€¦ · Amino acids, acylcarnitines, succinylacetone, dried blood spot, nonderivatized, derivatized, TSQ Endura Goal

Ap

plicatio

n N

ote 6

23

IntroductionFlow injection tandem mass spectrometry (FIA-MS/MS) has been frequently used for analysis of amino acids (AA) and acylcarnitines (AC) in dried blood spots for inborn errors of metabolism research.1-3 Established methods for detecting succinylacetone (SUAC) can be laborious because they require additional extraction due to its insolubility in the first extraction solution for AA and AC. In this application note, a single extraction step was used to simultaneously analyze AA, AC, and SUAC in dried blood spots on the Thermo Scientific™ TSQ Endura™ triple quadrupole mass spectrometer.4

The original sample preparation techniques use butyl esterification (i.e., derivatized) of amino acids and acylcarnitines in dried blood spots (DBS) due to the increased sensitivity that derivatization provides.

However, with the improved sensitivity of new mass spectrometry technologies, it is possible to detect both amino acids and acylcarnitines as their native free acids (i.e., nonderivatized). This simplifies analytical operation and minimizes the use of corrosive chemicals. In this application note, both nonderivatization and derivatization sample preparation methods were compared.

Many previous FIA-MS/MS studies on dried blood spots deployed neutral-loss scan mode (acylcarnitines) and precursor ion scan mode (some amino acids) for fast method development. In this application note, selected-reaction monitoring (SRM) was used for all AA, AC, and SUAC data acquisition. The advantage of SRM is that it accurately quantifies analytes and ensures both high selectivity and sensitivity, which especially benefited analysis of analytes that ionize poorly.

MethodSample PreparationSets of isotope-labeled internal standards of amino acids (NSK-A), acylcarnitines (NSK-B and NSK-B-G), and succinylacetone (NSK-T) were purchased from Cambridge Isotope Laboratories, Inc. The daily working internal standard concentration is listed in Table 1. Hydrazine, 1-butanol, and acetyl chloride were purchased from Sigma-Aldrich®. The other reagents were from Thermo Fisher Scientific.

The DBS quality control (QC) samples were kindly provided by the United States Centers for Disease Control and Prevention (CDC) for research purposes. The QC samples contained enriched analytes at three concentrations: low, intermediate, and high.

Key WordsAmino acids, acylcarnitines, succinylacetone, dried blood spot, nonderivatized, derivatized, TSQ Endura

GoalTo develop flow injection analysis-tandem mass spectrometry (FIA-MS/MS) research methods for simultaneous detection and quantification of 12 amino acids, 18 acylcarnitines, and succinylacetone in dried blood spots and compare derivatization and nonderivatization sample preparation methods.

Simultaneous Analysis of Amino Acids, Acylcarnitines, and Succinylacetone in Dried Blood Spots for Research Using Nonderivatized and Derivatized Methods Xiaolei Xie and Marta Kozak, Thermo Fisher Scientific, San Jose, CA

Key

Alanine (Ala) Valine (Val) Hydroxyisovalerylcarnitine (C5OH)

Arginine (Arg) Succinylacetone (SUAC) Hexanoylcarnitine (C6)

Aspartic acid (Asp) Free carnitine (C0) Octanoylcarnitine (C8)

Citrulline (Cit) Acetylcarnitine (C2) Decanoylcarnitine (C10)

Glutamic acid (Glu) Propionylcarnitine (C3) Dodecanoylcarnitine (C12)

Glycine (Gly) Malonylcarnitine (C3DC) Myristoylcarnitine (C14)

Leucine (Leu) Butyrylcarnitine (C4) Palmitoylcarnitine (C16)

Methionine (Met) Hydroxybutyrylcarnitine (C4OH) Hydroxypalmitoylcarnitine (C16OH)

Ornithine (Orn) Isovalerylcarnitine (C5) Stearoylcarnitine (C18)

Phenylalanine (Phe) Glytarylcarnitine (C5DC) Hydroxystearoylcarnitine (C18OH)

Tyrosine (Tyr)

2

The following protocols were used to prepare the DBS samples:

Derivatized1. Punch one 1/8 inch diameter disc from DBS sample into

a 96-well plate.

2. Add 100 µL of working internal standard solution (containing internal standards of 12 amino acids, 12 acylcarnitines, and SUAC) to each well.

3. Shake the well plate for 45 min at 45 °C.

4. Transfer the eluates to another well plate and evaporate at 50 °C under nitrogen flow.

5. Pipet 50 µL of methanol into each sample well and evaporate under nitrogen flow.

6. Pipet 50 µL of 3 n-butanol HCl into each sample well and incubate at 65 °C for 20 min. Then, evaporate under nitrogen flow.

7. Reconstitute each sample well with 100 µL of 50:50:0.02 acetonitrile/water/formic acid.

Nonderivatized1. Punch one 1/8 inch diameter disc from DBS sample into

a 96-well plate.

2. Add 100 µL of working internal standard solution (containing internal standards of 12 amino acids, 12 acylcarnitines, and SUAC) to each well.

3. Shake the well plate for 45 min at 45 °C.

4. Transfer the eluates to another well plate and evaporate at 50 °C under nitrogen flow.

5. Pipet 50 µL of methanol into each sample well and evaporate under nitrogen flow.

6. Reconstitute each sample well with 100 µL of 50:50:0.02 acetonitrile/water/formic acid.

Liquid Chromatography

Pump Thermo Scientific™ Dionex™ UltiMate™ HPG-3200 RS

Autosampler UltiMate WPS-3000 TRS

HPLC column None

Mobile phase 50:50:0.02 acetonitrile/water/formic acid

LC flow gradient Refer to Table 2

Mass SpectrometryFlow injection MS/MS analysis was performed on a TSQ Endura triple quadrupole mass spectrometer. The mass spectrometer conditions were as follows:

Ionization Heated electrospray ionization (HESI)

Spray voltage Positive, 3500 V

Sheath gas 50 Arb

Aux gas 7 Arb

Sweep gas 0 Arb

Ion transfer tube temperature 350 °C

Vaporizer temperature 200 °C

Data acquisition mode Selected-reaction monitoring (SRM)

Cycle time 1 s

Q1 resolution (FWHM) 0.7

Q3 resolution (FWHM) 0.7

CID gas 1.5 mTorr

Source fragmentation 0 V

Chrom filter 3 s

SRM parameters Refer to Table 3 (Derivatized) and Table 4 (Nonderivatized)

Internal Standard Concentrations (µmol/L)

Alanine-d4

2.50

Arginine-13C-d4

2.50

Aspartic acid-d3

2.50

Citrulline-d2

2.50

Glutamic acid-d3

2.50

Glycine-13C-15N 12.50

Leucine-d3

2.50

Methionine-d3

2.50

Ornithine-d2

2.50

Phenylalanine-13C6

2.50

Tyrosine-13C6

2.50

Valine-d8

2.50

Succinylacetone-13C5

2.50

C0-Carnitine-d9

0.76

C2-Carnitine-d3

0.19

C3-Carnitine-d3

0.04

C4-Carnitine-d3

0.04

C5-Carnitine-d9

0.04

C5DC-Carnitine-d3

0.08

C5OH-Carnitine-d3

0.04

C8-Carnitine-d3

0.04

C12-Carnitine-d9

0.04

C14-Carnitine-d9

0.04

C16-Carnitine-d3

0.08

C18-Carnitine-d3

0.08

Time (min) Flow Rate (mL/min) %A (mobile phase)

0.00 0.09 100

1.23 0.09 100

1.25 0.30 100

1.50 0.09 100

Table 1. Daily working internal standard concentrations.

Table 2. LC flow gradient.

3

Analyte Precursor Ion Product Ion Internal Standard Precursor

Ion Product Ion Collision Energy (V)

RF Lens (V)

Alanine 146.20 44.20 Alanine-d4

150.20 48.18 17 62

Arginine 231.28 70.13 Arginine-13C-d4

236.28 75.13 30 87

Aspartic acid 246.18 144.13 Aspartic acid-d3

249.25 147.13 15 103

Citrulline 232.28 113.13 Citrulline-d2

234.28 115.18 20 85

Glutamic acid 260.28 157.93 Glutamic acid-d3

263.33 161.13 16 94

Glycine 131.80 76.05 Glycine-13C-15N 134.20 78.10 8 55

Leucine 188.25 86.10 Leucine-d3

191.25 89.18 15 75

Methionine 206.23 104.13 Methionine-d3

209.20 107.23 16 81

Ornithine 189.25 70.10 Ornithine-d2

191.18 72.13 24 79

Phenylalanine 222.25 120.13 Phenylalanine-13C6

228.33 126.18 19 105

Tyrosine 238.30 136.13 Tyrosine-13C6

244.28 142.15 18 93

Valine 174.25 72.13 Valine-d8

182.23 80.18 16 73

SUAC 211.18 137.05 SUAC-13C5

216.18 142.05 12 91

C0-Carnitine 218.28 85.05 C0-Carnitine-d9

227.33 85.05 28 104


263.30 85.05 25 113

C3-Carnitine 274.33 85.05C3-Carnitine-d

3277.33 85.05 25 121

C3DC-Carnitine 360.33 85.05


3291.33 85.05 27 117

C4OH-Carnitine 304.33 85.05


9311.38 85.05 30 113

C6-Carnitine 316.35 85.05

C5DC-Carnitine 388.35 85.05 C5DC-Carnitine-d3

391.35 85.05 31 138

C5OH-Carnitine 318.38 85.05 C5OH-Carnitine-d3

321.38 85.05 31 138


3347.38 85.05 32 141

C10-Carnitine 372.40 85.05


409.43 85.05 36 184


437.48 85.05 35 193


3459.55 85.05 37 183



3487.55 85.05 38 215


Table 3. SRM parameters (derivatized).

4

Data ProcessingTandem MS data were processed using a new meta-calculation software, iRC PRO (2Next srl, Prato, Italy). This off-line automated data processing tool can process peak area, concentration, and user-defined formulas (Figure 1).

The metacalculation software improves time effectiveness by eliminating the manual calculation process and removing transcription errors in the post-analytical phase. The processing time is reduced from hours to minutes.

Assay ValidationThe intra-assay precision was determined at three concentrations by means of ten successive, independent measurement of DBS samples (n=10). The inter-assay precision was determined at three concentrations by means of ten independent measurement of DBS samples in seven different test series (n=70).

Analyte Precursor Ion Product Ion Internal Standard Precursor

Ion Product Ion Collision Energy (V)

RF Lens (V)

Alanine 90.15 44.20 Alanine-d4

94.15 48.20 13 45

Arginine 175.23 70.15 Arginine-13C-d4

180.23 75.15 24 92

Aspartic acid 134.20 116.13 Aspartic acid-d3

137.20 119.13 6 54

Citrulline 176.20 113.13 Citrulline-d2

178.20 115.13 18 59

Glutamic acid 148.15 130.08 Glutamic acid-d3

151.15 133.08 9 62

Glycine 76.08 30.25 Glycine-13C-15N 78.08 32.25 13 43

Leucine 132.25 86.13 Leucine-d3

135.25 89.13 11 56

Methionine 150.18 133.08 Methionine-d3

153.18 136.08 9 60

Ornithine 133.15 70.15 Ornithine-d2

135.15 72.15 19 63

Phenylalanine 166.20 120.15 Phenylalanine-13C6

172.20 126.15 16 69

Tyrosine 182.15 136.18 Tyrosine-13C6

188.15 142.18 15 71

Valine 118.23 72.15 Valine-d8

126.23 80.15 13 53

SUAC 155.18 109.12 SUAC-13C5

160.18 114.12 22 63


171.23 85.05 23 69


207.23 85.05 21 96


3221.23 85.05 23 91

C3DC-Carnitine 248.23 85.05


3235.18 85.05 21 78



9255.30 85.05 25 96

C6-Carnitine 260.30 85.05

C5DC-Carnitine 276.30 85.05 C5DC-Carnitine-d3

279.30 85.05 25 96

C5OH-Carnitine 262.30 85.05 C5OH-Carnitine-d3

265.30 85.05 25 96


3291.33 85.05 26 108

C10-Carnitine 316.33 85.05


353.45 85.05 39 152


381.45 85.05 39 152


3403.45 85.05 36 185



3431.45 85.05 36 185


Table 4. SRM parameters (nonderivatized).

Figure 1. iRC PRO intuitive workflow – icon-based user interface.

5ResultsThe derivatization process using butanol converted free amino acids and acylcarnitines into the butyric esters and added a mass of 56 (except for aspartic acid, glutamic acid, and C5DC, in which a mass of 112 was added). Figure 2 and 3 show full-scan spectra of derivatized and nonderivatized internal standards respectively.

Figure 2. Full-scan spectra of derivatized internal standards.

0 5

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

100

Rel

ativ

e A

bund

ance

216.20 263.23

150.20 134.09

459.47

487.46 191.22 311.23 409.47

391.36 437.46 347.33

291.28 249.23

277.28

244.22

321.27

227.31

228.27

234.22 236.22

209.23

182.17

100 150 200 250 300 350 400 450 500 m/z

Internal Standard m/z Internal Standard m/z

Alanine-d4

150.20 C0-Carnitine-d9

227.31

Arginine-13C-d4


263.23

Aspartic acid-d3


277.28

Citrulline-d2


291.28

Glutamic acid-d3


311.23

Glycine-13C-15N 134.09 C5DC-Carnitine-d3

391.36

Leucine-d3

191.22 C5OH-Carnitine-d3

321.27

Methionine-d3


347.33

Ornithine-d2


409.47

Phenylalanine-13C6


437.46

Tyrosine-13C6


459.47

Valine-d8


487.46


216.20

6

SRM was used to acquire MS/MS data for all the analytes. Collision energy and RF lens parameters were optimized for each target and internal standard to ensure maximum selectivity and sensitivity. SRM allowed acquisition of peaks with good signal-to-noise ratios even for analytes with poor ionization such as SUAC and C5DC regardless of whether derivatization was used (Figure 4 and 5).

Figure 3. Full-scan spectra of nonderivatized internal standards.

100 150 200 250 300 350 400 450 500 m/z

0 5

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

100

Rel

ativ

e A

bund

ance

78.07

171.24 431.42

135.19 160.18

403.41 221.18 381.42

188.16

207.21

94.07 291.29 353.36

255.27 137.20

151.15

235.21 126.17

153.12

172.20

180.22

178.14

279.14

265.24

Internal Standard m/z Internal Standard m/z

Alanine-d4


171.24

Arginine-13C-d4


207.21

Aspartic acid-d3


221.18

Citrulline-d2


235.21

Glutamic acid-d3


255.27

Glycine-13C-15N 78.07 C5DC-Carnitine-d3

279.14

Leucine-d3

135.19 C5OH-Carnitine-d3

265.24

Methionine-d3


291.29

Ornithine-d2


353.36

Phenylalanine-13C6


381.42

Tyrosine-13C6


403.41

Valine-d8


431.42


160.18

7

Intra-assay PrecisionFor the derivatized method, the average intra-assay precisions (n=10) for 12 AA and SUAC at three concentrations were 7.9% (low), 8.0% (intermediate), and 8.0% (high). The average intra-assay precisions for 18 AC at three concentrations were 8.9% (low), 8.3% (intermediate), and 9.0% (high) (Table 5).

For the nonderivatized method, the average intra-assay precisions (n=10) for AA and SUAC at three concentrations were 6.1% (low), 7.2% (intermediate), and 9.8% (high). The average intra-assay precisions for AC at three concentrations were 7.6% (low), 6.2% (intermediate), and 8.2% (high) (Table 6).

Figure 4. Flow injection analysis (FIA) profiles of SUAC-13C5, SUAC and C5DC-D3, C5DC using derivatized method.

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4Time (min)

0

50000

100000

150000

200000

2500000

500000

1000000

1500000

2000000

2500000In

tens

ity

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4Time (min)

0

5000

10000

0

2000

4000

6000

8000

10000

Inte

nsity

SUAC-13C5

SUAC

C5DC-d3

C5DC

SUAC-13C5

SUAC

C5DC-d3

C5DC

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4Time (min)

0

2000

4000

6000

80000

20000

40000

60000

80000

Inte

nsity

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4Time (min)

0

500

1000

1500

2000

25000

500

1000

1500

Inte

nsity

Figure 5. Flow injection analysis (FIA) profiles of SUAC-13C5, SUAC and C5DC-D3, C5DC using nonderivatized method.

8

AnalyteCoefficient of Variation (%) Concentrations in µmol/L

Low Intermediate High Low Intermediate High

Alanine 9.5 8.9 11.5 552.3 710.2 890.3

Arginine 5.4 9.6 7.6 111.2 211.4 317.8

Aspartic acid 9.0 6.5 8.0 N/A N/A N/A

Citrulline 6.8 4.0 5.6 55.8 131.6 277.4

Glutamic acid 10.1 5.9 4.7 N/A N/A N/A

Glycine 8.8 8.2 6.8 N/A N/A N/A

Leucine 8.5 8.4 6.8 225.1 378.8 633.0

Methionine 7.7 6.4 8.7 65.1 154.6 257.5

Ornithine 8.4 12.3 8.4 N/A N/A N/A

Phenylalanine 7.5 8.5 5.5 169.8 274.9 369.7

Tyrosine 7.8 10.8 8.6 236.6 416.5 605.0

Valine 9.6 8.3 8.1 289.5 410.0 547.8

SUAC 8.2 7.2 9.4 1.9 4.6 9.9

C0-Carnitine 12.2 5.0 6.8 35.3 54.4 70.6

C2-Carnitine 10.8 8.6 7.6 24.6 37.6 47.9

C3-Carnitine 11.6 12.7 11.7 4.9 10.1 14.6

C3DC-Carnitine 7.4 6.9 9.0 0.2 0.6 1.1

C4-Carnitine 6.7 6.5 10.6 1.1 2.8 5.0

C4OH-Carnitine 7.1 6.1 8.6 0.3 0.6 1.4

C5-Carnitine 6.9 5.4 10.6 0.6 1.7 3.2

C6-Carnitine 8.1 5.7 5.9 0.7 1.3 3.3

C5DC-Carnitine 4.7 8.6 8.3 1.0 2.2 3.1

C5OH-Carnitine 8.4 7.3 9.1 0.6 1.2 2.8

C8-Carnitine 9.0 9.5 4.0 0.6 1.2 2.7

C10-Carnitine 7.4 6.8 6.9 0.6 1.2 3.1

C12-Carnitine 6.1 6.7 8.8 0.7 1.3 2.8

C14-Carnitine 8.9 10.8 8.7 0.6 1.5 2.9

C16-Carnitine 10.7 10.9 10.8 3.5 7.8 11.8

C16OH-Carnitine 12.6 13.0 12.6 0.1 0.4 0.7

C18-Carnitine 10.2 7.4 13.6 1.6 2.6 5.6

C18OH-Carnitine 10.9 11.1 8.8 0.4 0.7 1.1

Table 5. Derivatized method intra-assay precision at three concentrations (low, intermediate, and high). n=10.

N/A, the analytes were not enriched in QC samples.

9

Inter-assay PrecisionFor the derivatized method, the average inter-assay precisions (n=70) for 12 AA and SUAC at three concentrations were 13.5% (low), 12.9% (intermediate), and 12.5% (high). The average inter-assay precisions for 18 AC at three concentrations were 15.0% (low), 15.6% (intermediate), and 16.1% (high) (Table 7).

For the nonderivatized method, the average inter-assay precisions (n=70) for AA and SUAC at three concentrations were 12.8% (low), 12.8% (intermediate), and 12.6% (high). The average inter-assay precisions for AC at three concentrations were 12.7% (low), 10.5% (intermediate), and 11.8% (high) (Table 8).



Alanine 4.7 7.2 11.7 531.2 749.2 894.0

Arginine 6.1 7.2 9.7 104.8 206.2 303.3


Citrulline 4.4 7.8 8.0 54.0 127.3 269.0


Glycine 8.6 9.7 10.6 N/A N/A N/A

Leucine 5.5 6.3 9.2 252.9 416.0 637.4

Methionine 8.1 4.8 9.7 65.0 155.7 253.7


Phenylalanine 4.9 5.7 9.4 164.1 266.2 347.7

Tyrosine 5.2 5.9 7.5 240.4 431.7 623.5

Valine 5.1 6.3 10.1 295.9 439.5 547.3

SUAC 10.5 14.1 13.0 1.9 4.2 9.1

C0-Carnitine 5.6 6.0 6.6 30.8 43.4 56.9

C2-Carnitine 6.7 5.4 6.8 24.1 38.3 48.5

C3-Carnitine 8.7 3.9 8.9 4.6 10.1 14.6

C3DC-Carnitine 6.9 6.5 5.9 0.3 0.6 1.4

C4-Carnitine 9.6 5.2 8.5 1.1 2.8 5.3

C4OH-Carnitine 5.2 5.5 7.3 0.3 0.7 1.4

C5-Carnitine 7.8 7.3 9.0 0.6 1.7 3.2

C6-Carnitine 6.3 6.8 10.8 0.7 1.4 3.3

C5DC-Carnitine 8.7 7.1 10.3 1.1 1.9 2.7

C5OH-Carnitine 10.1 7.8 10.3 0.5 1.0 2.3

C8-Carnitine 8.3 5.2 7.8 0.6 1.1 2.7

C10-Carnitine 9.6 6.8 9.2 0.8 1.6 3.9

C12-Carnitine 6.7 4.6 6.5 0.4 0.9 2.1

C14-Carnitine 5.8 8.2 5.9 0.5 1.4 2.6

C16-Carnitine 7.8 4.0 5.5 3.7 8.9 12.2

C16OH-Carnitine 5.9 8.3 8.7 0.1 0.4 0.7

C18-Carnitine 7.1 3.4 9.5 1.7 2.7 5.6

C18OH-Carnitine 9.6 10.1 10.4 0.4 0.8 1.4

Table 6. Nonderivatized method intra-assay precision at three concentrations (low, intermediate, and high). n=10.


10



Alanine 12.2 9.6 10.3 538.1 711.4 882.2

Arginine 17.1 16.6 18.6 123.8 222.5 326.0


Citrulline 17.0 14.8 12.5 58.9 132.6 285.3


Glycine 10.3 12.2 10.4 N/A N/A N/A

Leucine 12.2 12.2 12.1 224.2 381.2 640.3

Methionine 13.2 11.6 11.5 66.2 154.5 246.3


Phenylalanine 11.8 12.8 12.5 172.1 271.6 362.2

Tyrosine 13.6 12.5 14.0 232.9 400.9 604.1

Valine 11.4 12.8 11.5 290.4 409.3 539.3

SUAC 13.0 13.4 9.4 2.0 4.7 9.9

C0-Carnitine 15.7 15.1 13.4 36.4 51.7 71.8

C2-Carnitine 13.8 14.1 15.1 24.2 37.1 49.4

C3-Carnitine 16.3 15.3 16.6 4.8 9.5 14.2

C3DC-Carnitine 13.8 14.1 16.1 0.2 0.5 1

C4-Carnitine 16.3 13.4 17.5 1.0 2.5 4.7

C4OH-Carnitine 16.2 18.4 15.5 0.3 0.6 1.3

C5-Carnitine 15.1 14.6 16.6 0.6 1.5 2.9

C6-Carnitine 14.9 12.3 14.2 0.7 1.3 3.1

C5DC-Carnitine 13.7 15.6 15.2 1.1 2.1 3.1

C5OH-Carnitine 13.4 16.2 14.7 0.6 1.1 2.7

C8-Carnitine 15.4 13.8 16.6 0.6 1.1 2.6

C10-Carnitine 17.4 16.9 18.0 0.6 1.1 2.8

C12-Carnitine 15.4 17.1 17.3 0.7 1.2 2.6

C14-Carnitine 14.5 14.9 17.1 0.6 1.4 2.7

C16-Carnitine 14.7 16.1 16.2 3.7 7.9 11.5

C16OH-Carnitine 13.0 17.6 18.3 0.1 0.4 0.7

C18-Carnitine 14.7 18.2 15.5 1.7 2.5 5.1

C18OH-Carnitine 15.2 17.1 16.5 0.4 0.7 1.1

Table 7. Derivatized method inter-assay precision at three concentrations (low, intermediate, and high). n=70.


11

Method ComparisonThe concentration of analytes obtained from nonderivatized and derivatized methods were compared. The average method differences of 12 AA and SUAC between quantitative values resulting from derivatization and nonderivatization methods at three concentrations were 3.8% (low), 4.8% (intermediate), and 3.2% (high). The average method differences of 18 AC at three concentrations were 14.2% (low), 11.4% (intermediate), and 10.5% (high) (Figure 6). Therefore the two methods were highly correlated. Our data are consistent with the reported results from a comprehensive empirical analysis.5



Alanine 20.0 16.1 15.6 632.1 863.2 1002.4

Arginine 12.0 11.3 12.2 103.3 204.2 303.2


Citrulline 10.7 11.4 9.7 54.3 129.0 263.3


Glycine 13.4 13.7 14.8 N/A N/A N/A

Leucine 10.8 9.7 10.2 260.6 414.2 646.0

Methionine 18.8 17.5 20.2 53.8 129.2 200.1


Phenylalanine 7.7 8.7 11.2 162.9 268.8 351.3

Tyrosine 8.1 10.0 10.8 239.7 435.9 615.8

Valine 9.1 9.3 10.1 300.1 432.9 539.5

SUAC 18.1 21.0 13.7 2.7 4.9 9.3

C0-Carnitine 12.5 11.3 12.0 29.4 40.3 51.9

C2-Carnitine 10.3 10.0 10.9 24.1 38.1 47.8

C3-Carnitine 9.8 9.7 11.8 4.5 9.9 14.3

C3DC-Carnitine 12.4 11.8 9.1 0.3 0.6 1.3

C4-Carnitine 10.3 10.8 11.6 1.1 2.7 5.2

C4OH-Carnitine 11.3 10.5 10.6 0.3 0.6 1.4

C5-Carnitine 11.2 11.6 11.6 0.6 1.7 3.2

C6-Carnitine 16.9 16.5 12.7 0.7 1.4 3.2

C5DC-Carnitine 11.3 9.1 10.1 1.0 1.9 2.6

C5OH-Carnitine 12.8 11.3 12.3 0.5 1.0 2.4

C8-Carnitine 9.9 8.6 10.7 0.6 1.1 2.6

C10-Carnitine 18.4 13.5 13.2 0.8 1.6 4.0

C12-Carnitine 12.2 8.7 9.8 0.4 0.9 2.1

C14-Carnitine 11.3 8.0 10.0 0.5 1.4 2.7

C16-Carnitine 10.9 8.4 12.2 3.6 8.7 12.2

C16OH-Carnitine 14.3 9.6 13.9 0.1 0.4 0.7

C18-Carnitine 12.1 7.8 11.6 1.7 2.7 5.7

C18OH-Carnitine 21.3 11.2 17.8 0.4 0.9 1.5

Table 8. Nonderivatized method inter-assay precision at three concentrations (low, intermediate, and high). n=70.

Figure 6. Comparisons between quantitative values of 12 AA, SUAC, and 18 AC resulting from nonderivatized and derivatized methods.

0

100

200

300

400

500

600

700

800

900

1000

0 100 200 300 400 500 600 700 800 900 1000

Der

ivat

ized

(m

ol/L

)

Nonderivatized ( mol/L)

Low Intermediate High

0

20

40

60

0 20 40 60


Ap

plicatio

n N

ote 6

23

AN64265-EN 0616S

Conclusion• Flow injection-tandem mass spectrometry methods

were developed to simultaneously detect and quantify amino acids, acylcarnitines, and succinylacetone in a single extraction process in dried blood spots for research. Rapid data processing was performed using iRC Pro metacalculation software.

• Both derivatization and nonderivatization sample preparation methods were capable of accurately quantifying AA/AC/SUAC on TSQ Endura triple quadrupole MS with a run time of 1.5 min.

• SRM data acquisition mode optimized for each analyte and internal standard guarantees both high sensitivity and high selectivity.

• The TSQ Endura MS system can provide average intra-assay precision (n=10) at three enriched concentrations of less than 10% and average inter-assay precision (n=70) of less than 15% for both nonderivatized and derivatized methods.

• The method difference between quantitative values resulting from nonderivatized and derivatized methods was minor and both methods are highly correlated.

References1. Chace, D.H.; Kalas, T.A.; Naylor, E.W. Use of tandem

mass spectrometry for multianalyte screening of dried blood specimens from newborns. Clin. Chem., 2003, 49(11), 1797-817.

2. Chace, D.H., et al. Rapid diagnosis of phenylketonuria by quantitative analysis for phenylalanine and tyrosine in neonatal blood spots by tandem mass spectrometry. Clin. Chem., 1993, 39(1), 66-71.

3. Millington, D.S., et al. Tandem mass spectrometry: a new method for acylcarnitine profiling with potential for neonatal screening for inborn errors of metabolism. J. Inherit. Metab. Dis., 1990, 13(3), 321-4.

4. Dhillon, K.S., et al. Improved tandem mass spectrometry (MS/MS) derivatized method for the detection of tyrosinemia type I, amino acids and acylcarnitine disorders using a single extraction process. Clin. Chim. Acta, 2011, 412(11-12), 873-9.

5. De Jesus, V.R., et al. Comparison of amino acids and acylcarnitines assay methods used in newborn screening assays by tandem mass spectrometry. Clin. Chim. Acta, 2010, 411(9-10), 684-9.

For research use only. Not for use in diagnostic procedures.

Africa +43 1 333 50 34 0Australia +61 3 9757 4300Austria +43 810 282 206Belgium +32 53 73 42 41Canada +1 800 530 8447China 800 810 5118 (free call domestic)

400 650 5118

Denmark +45 70 23 62 60Europe-Other +43 1 333 50 34 0Finland +358 9 3291 0200France +33 1 60 92 48 00Germany +49 6103 408 1014India +91 22 6742 9494Italy +39 02 950 591

Japan +81 45 453 9100Korea +82 2 3420 8600Latin America +1 561 688 8700Middle East +43 1 333 50 34 0Netherlands +31 76 579 55 55New Zealand +64 9 980 6700Norway +46 8 556 468 00

Russia/CIS +43 1 333 50 34 0Singapore +65 6289 1190Spain +34 914 845 965Sweden +46 8 556 468 00Switzerland +41 61 716 77 00UK +44 1442 233555USA +1 800 532 4752

www.thermofisher.com©2016 Thermo Fisher Scientific Inc. All rights reserved. Sigma-Aldrich is a registered trademark of Sigma-Aldrich Co. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.

Documents

Simultaneous Analysis of Amino Acids, Acylcarnitines, and ...€¦ · Amino acids, acylcarnitines, succinylacetone, dried blood spot, nonderivatized, derivatized, TSQ Endura Goal